HIV infection is associated with persistent, aberrant immune activation that supports viral replication and contributes to the progressive immune dysfunction associated with HIV disease progression. Chronic immune activation, whether induced by persistent foreign antigens or loss of self tolerance, activates and expands host-mediated immunosuppressive mechanisms that serve to prevent immune-mediated damage to the host. In addition to limiting autoimmunity, several of these mechanisms suppress immune responses directed against foreign antigens and affect the ability of the host to eliminate or control certain tumors andbacterial, parasitic and viral infections. Two of these mechanisms in particular, suppressor CD25+ CD4+FoxP3+ regulatory T (Treg) cells and the negative regulatory surface molecule programmed death (PD)-1, play a role in suppressing virus-specific T cell responses in chronic viral infection and/or vaccination. [unreadable] [unreadable] We have demonstrated that CD25+ Treg cells isolated from peripheral blood (PB) of asymptomatic HIV+ subjects inhibit numerous HIV-specific CD4+ and CD8+ T cell immune responses in vitro, including proliferation, cytokine and chemokine secretion, and the ability of CD8+ T cells to kill HIV-expressing target cells. It remains controversial whether CD25+CD4+ Treg cells are preferentially lost with HIV disease progression and whether this loss may be a contributing factor in hyperimmune activation associated with untreated progressive HIV infection. Phenotypic studies quantifying Treg cells in peripheral blood (PB) versus tissue suggest that Treg cells are not lost, but instead migrate from PB to lymphoid tissue (LT) sites of HIV antigen expression during periods of viral replication. However, the suppressive capacity of LT associated Treg cells isolated from HIV+ individuals has not been established. We have recently demonstrated that functional CD25+ Treg cells, capable of suppressing HIV-specific cytolytic activity, are present in the LT of HIV+ subjects, including those with advanced disease (high VL and low CD4+ T cell counts). Treg-mediated immunosuppression in LT may be particularly relevant as this is the primary site of adaptive immune response priming as well as a major site of HIV replication. Of interest, Treg cells capable of inhibiting HIV-specific cytolytic activity, but not proliferation, were detected also in the blood of HIV+ subjects with advanced disease, although they were less potent than those isolated from LT. These results support the concept that at least certain Treg subsets are maintained, particularly in the LT, throughout HIV disease. Finally, we have continued to investigate the role of Treg cells in non-pathogenic HIV animal models. SIV-infected sootey mangabeys (SM) usually do not show signs of disease despite high levels of SIV replication. Lack of disease in SM correlates with significantly lower levels of immune activation compared to macaques that succumb to pathogenic SIV infection. In collaborative studies, we found that high numbers of CD25+ Treg cells in SM correlated with high CD4+ T counts, an indicator of a disease-free state. [unreadable] [unreadable] We have also initiated studies investigating the role of Programmed Death (PD)-1 and its ligands PD-L1 and PD-L2, negative regulatory members of the B7 superfamily, in the functional exhaustion of HIV-specific T cells. Engagement of PD-1 has been shown to suppress T cell receptor (TCR)-mediated signaling events and has been associated with increased apoptosis. PD-1 is elevated in HIV-specific T cells and interference with the interaction between PD-1 and PD-L1, alone or in combination with PD-L2, rescues numerous functions of exhausted HIV-specific T cells in vitro. The ability to restore the quality (broad functionality), and not just the quantity, of HIV-specific T cells has been a primary goal of immune-based therapies for the treatment of HIV disease. However, controversy exists regarding whether blocking PD-1 activation increases functionality of HIV-specific T cells on a per-cell basis or simply prevents the apoptosis of PD-1+ HIV-specific T cells following stimulation, thus allowing for increased expansion of existing HIV-specific T cells.[unreadable] [unreadable] We observed that treatment of PB mononuclear cells (PBMCs) from HIV+ subjects in vitro with neutralizing anti-PD-L1, but not anti-PD-L2, antibodies significantly enhances HIV-specific T cell proliferative responses. In contrast, in vitro treatment of PBMCs with neutralizing anti-PD-L2, but not anti-PD-L1, antibodies modestly reduced apoptosis of PD-1+ T cells. Both PD-L1 and PD-L2 antibodies failed to rescue cytokine production following a short stimulation (6h) of PBMCs with HIV peptides, although exogenous PD-L1 or PD-L2 effectively suppressed anti-CD3/CD28-stimulated cytokine production. These data point to potentially differential roles/mechanisms of endogenous PD-L1 and PD-L2 in the suppression of HIV-specific T cell proliferation and survival, respectively. Furthermore, these data suggest that PD-L1 and PD-L2, as expressed on cells in the blood, do not directly inhibit the effector function (cytokine production) of PD-1+ HIV-specific T cells on a per-cell basis. However, we observed that both PD-1 and PD-1 ligand expression was greater in LT than in PB and that PD-1 ligand expression in the LT, but not the PB, correlated with markers of HIV disease progression. It remains to be established whether the quality of HIV-specific T cell function can be enhanced by blocking PD-1 and PD-1 ligand interactions in the LT.[unreadable] [unreadable] Finally, we have investigated the role of the PD-1 axis in the context of TCR-independent, cytokine-driven T cell activation. We tested numerous pro-inflammatory, anti-inflammatory and immunoregulatory cytokines for the ability to modulate the expression of PD-1 and its ligands in vitro. Of those cytokines tested, only IL-2, IL-7 and IL-15 were found to directly upregulate PD-1 on purified T cells, an effect that correlated with the ability to directly activate and induce proliferation of T cells PD-1 expression on both naive and memory T cell subsets was increased by cytokines (this sentence doesn't make sense). Furthermore, these cytokines upregulated the expression of PD-L1 and PD-L2 on monocytes in the context of total PBMCs. In support of these observations, significant increases the frequency of PD-1+ T cells and PD-L1+ and PD-L2+ monocytes were observed in PBMCs of HIV+ individuals following administration of IL-2 in vivo. IL-7 and IL-15, and to a lesser degree, IL-2, play an important role in T cell homeostasis, supporting the survival and expansion of both naive and memory T cells and thus elevated PD-1 expression could have considerable functional consequences.